The present invention relates to a compound. In addition, the present invention relates to processes for making the compound and to the use of that compound in therapy, in particular gene therapy (especially gene transfer).
One aspect of gene therapy involves the introduction of foreign nucleic acid (such as DNA) into cells, so that its expressed protein may carry out a desired therapeutic function.1 
Examples of this type of therapy include the insertion of TK, TSG or ILG genes to treat cancer; the insertion of the CFTR gene to treat cystic fibrosis; the insertion of NGF, TH or LDL genes to treat neurodegenerative and cardiovascular disorders; the insertion of the IL-1 antagonist gene to treat rheumatoid arthritis; the insertion of HIV antigens and the TK gene to treat AIDS and CMV infections; the insertion of antigens and cytokines to act as vaccines; and the insertion of xcex2-globin to treat haemoglobinopathic conditions, such as thalassaemias.
Many current gene therapy studies utilise adenoviral gene vectorsxe2x80x94such as Ad3 or Ad5xe2x80x94or other gene vectors. However, serious problems have been associated with their use.2 This has prompted the development of less hazardous, non-viral approaches to gene transfer.3 
A non-viral transfer system of great potential involves the use of cationic liposomes.4 In this regard, cationic liposomesxe2x80x94which usually consist of a neutral phospholipid and a cationic lipidxe2x80x94have been used to transfer DNA4, mRNA5, antisense oligonucleotides6, proteins7, and drugs8 into cells. A number of cationic liposomes are commercially available4,9 and many new cationic lipids have recently been synthesised10. The efficacy of these liposomes has been illustrated by both in vitro4 and in vivo11.
A neutral phospholipid useful in the preparation of a cationic liposome is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium chloride, otherwise known as xe2x80x9cDOTMAxe2x80x9d. The structure of DOTMA is shown in FIG. 1.
One of the most commonly used cationic liposome systems consists of a mixture of a neutral phospholipid dioleoylphosphatidylethanolamine (commonly known as xe2x80x9cDOPExe2x80x9d) and a cationic lipid, 3xcex2-[(N,N-dimethylaminoethyl)carbamoyl]cholesterol (commonly known as xe2x80x9cDC-Cholxe2x80x9d)12. The structure of DOPE is shown in FIG. 2. The structure of DC-Chol is shown in FIG. 3.
A lipid has been synthesised by reacting spermidine and cholesterol chloroformate in CH2Cl2 in the presence of N,N-diisoprophylethylamine18. However, this resulted in a mixture of the lipid and the corresponding regio-isomeric lipid, which mixture proved inseparable by chromatography.
Despite the efficacy of the known cationic liposomes there is still a need to optimise the gene transfer efficiency of cationic liposomes in human gene therapy10.
According to one aspect of the present invention there is provided a compound capable of acting as a cationic lipid, the compound comprising a cholesterol group having linked thereto a head group; and wherein the head group is more positive than the head group of DC-Chol; but wherein the compound is not synthesised by reacting spermidine and cholesterol chloroformate in CH2Cl2 in the presence of N,N-diisoprophylethylamine.
As indicated above, the head group of DC-Chol is Me2N(CH2)2NHxe2x80x94.
According to another aspect of the present invention there is provided a process of preparing a compound according to the present invention comprising reacting a cholesterol group with a head group.
According to another aspect of the present invention there is provided a compound according to the present invention or a compound when prepared by the process of the present invention for use in therapy.
According to another aspect of the present invention there is provided the use of a compound according to the present invention or a compound when prepared by the process of the present invention in the manufacture of a medicament for the treatment of a genetic disorder or a condition or a disease.
According to another aspect of the present invention there is provided a cationic liposome formed from the compound according to the present invention or a compound when prepared by the process of the present invention.
According to another aspect of the present invention there is provided a method of preparing a cationic liposome comprising forming the cationic liposome from the compound according to the present invention or a compound when prepared by the process of the present invention.
According to another aspect of the present invention there is provided a cationic liposome according to the present invention or a cationic liposome as prepared by the method of the present invention for use in therapy.
According to another aspect of the present invention there is provided the use of a cationic liposome according to the present invention or a cationic liposome as prepared by the method of the present invention in the manufacture of a medicament for the treatment of genetic disorder or condition or disease.
According to another aspect of the present invention there is provided a combination of a nucleotide sequence and any one or more of: a compound according to the present invention, a compound when prepared by the process of the present invention, a liposome of the present invention, or a liposome as prepared by the method of the present invention.
According to another aspect of the present invention there is provided a combination according to the present invention for use in therapy.
According to another aspect of the present invention there is provided the use of a combination according to the present invention in the manufacture of a medicament for the treatment of genetic disorder or condition or disease.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising a compound according to the present invention or a compound when prepared by the process of the present invention admixed with a pharmaceutical and, optionally, admixed with a pharmaceutically acceptable diluent, carrier or excipient.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising a cationic liposome according to the present invention or a cationic liposome as prepared by the method of the present invention admixed with a pharmaceutical and, optionally, admixed with a pharmaceutically acceptable diluent, carrier or excipient.
It is believed that a key advantage of the compound of the present invention is that it can be used as a cationic lipid (amphiphile) in the preparation of a cationic liposome useful in gene therapy, in particular the transfer of nucleic acids (including genes and antisense DNA/RNA) into cells (in vitro and in vivo) to derive a therapeutic benefit.
The cholesterol group can be cholesterol or a derivative thereof. Examples of cholesterol derivatives include substituted derivatives wherein one or more of the cyclic CH2 or CH groups and/or one or more of the straight-chain CH2 or CH groups is/are appropriately substituted. Alternatively, or in addition, one or more of the cyclic groups and/or one or more of the straight-chain groups may be unsaturated.
In a preferred embodiment the cholesterol group is cholesterol. It is believed that cholesterol is advantageous as it stabilises the resultant liposomal bilayer.
Preferably the cholesterol group is linked to the head group via a carbamoyl linkage. It is believed that this linkage is advantageous as the resultant liposome has a low or minimal cytotoxicity.
Preferably the head group is a polyamine group. It is believed that the polyamine group is advantageous because it increases the DNA binding ability and efficiency of gene transfer of the resultant liposome.
In one embodiment, preferably the polyamine group is a naturally occurring polyamine. It is believed that the polyamine head-group is advantageous because the increased amino functionality increases the overall positive charge of the liposome. In addition, polyamines are known to both strongly bind and stabilise DNA14. In addition, polyamines occur naturally in cells and so it is believed that toxicological problems are minimised15.
Typical examples of suitable polyamines include spermidine, spermine, caldopentamine, norspermidine and norspermine. These polyamines are shown in FIG. 4.
Preferably the polyamine is spermidine or spermine as these polyamines are known to interact with single or double stranded DNA. An alternative preferred polyamine is caldopentamine.
Thus, a preferred compound is spermidine linked to cholesterol via a carbamate linkage. This compound is shown in FIG. 5. It is believed that the polyamino head group is advantageous for DNA condensation, the carbamate linkage is stable but biodegradable and the cholesteryl group imparts bilayer rigidity. The carbamate linkage may be part of, or an integral component of, the head group.
Another preferred compound is spermine linked to cholesterol via a carbamate linkage. Likewise, it is believed that the polyamino head group is advantageous for DNA condensation, the carbamate linkage is stable but biodegradable and the cholesteryl group imparts bilayer rigidity.
Preferably the compound is in admixture with or associated with a nucleotide sequence.
The nucleotide sequence may be part or all of an expression system that may be useful in therapy, such as gene therapy.
Preferably the process comprises at least one step utilising aza-Wittig methodology.
Preferably the process comprises the use of trimethylphosphine.
Preferably the process comprises the use of a molecular sieve.
Preferably, the cationic liposome is formed from the compound of the present invention and a neutral phospholipidxe2x80x94such as DOTMA or DOPE. Preferably, the neutral phospholipid is DOPE.
In another embodiment, preferably two or more of the amine groups of the polyamine group of the present invention are separated by one or more groups which are not found in nature that separate amine groups of naturally occuring polyamine compounds (i.e. preferably the polyamine group of the present invention has un-natural spacing).
In summation, the present invention provides a compound capable of acting as a cationic lipid, the compound comprising a cholesterol group having linked thereto a head group; and wherein the head group is more positive than the head group of DC-Chol; but wherein the compound is not synthesised by reacting spermidine and cholesterol chloroformate in CH2Cl2 in the presence of N,N-diisoprophylethylamine.
A preferred embodiment of the present invention is a compound capable of acting as a cationic lipid, the compound comprising a cholesterol group having linked thereto a head group; wherein the head group is more positive than the head group of DC-Chol; wherein the cholesterol group is cholesterol; and wherein the head group is a polyamine group; but wherein the compound is not synthesised by reacting spermidine and cholesterol chloroformate in CH2Cl2 in the presence of N,N-diisoprophylethylamine.
A more preferred embodiment of the present invention is a compound capable of acting as a cationic lipid, the compound comprising a cholesterol group having linked thereto a head group; wherein the head group is more positive than the head group of DC-Chol; wherein the cholesterol group is cholesterol; wherein the head group is a polyamine group; and wherein the cholesterol group is linked to the head group via a carbamoyl linkage; but wherein the compound is not synthesised by reacting spermidine and cholesterol chloroformate in CH2Cl2 in the presence of N,N-diisoprophylethylamine.
A highly preferred embodiment of the present invention is a compound capable of acting as a cationic lipid, the compound comprising a cholesterol group having linked thereto a head group; wherein the head group is more positive than the head group of DC-Chol; wherein the cholesterol group is cholesterol; wherein the head group is a polyamine group; wherein the cholesterol group is linked to the head group via a carbamoyl linkage, and wherein the polyamine group is a naturally occurring polyamine, such as any one of spermidine, spermine or caldopentamine; but wherein the compound is not synthesised by reacting spermidine and cholesterol chloroformate in CH2Cl2 in the presence of N,N-diisoprophylethylamine.